Crosslinking is the process of joining two or more molecules by a covalent bond. Crosslinking reagents contain reactive ends to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules. Because of the availability of several chemical groups in proteins and peptides that may be targets for reactions, proteins and peptides are readily conjugated and otherwise studied using crosslinking methods.
The traditional way to couple two proteins, for example through an amine (—NH2) functional group on one protein and a sulfhydryl (—SH) group on the other, is by using a heterobifunctional crosslinker that contains a N-hydroxysuccinimide (NHS) ester and a maleimide group, e.g. SMCC. NHS esters react with primary amines at pH 7-9 to form covalent amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. The traditional (above mentioned) heterobifunctional crosslinking strategy using SMCC is outlined in FIG. 1.
Although these reagents have been used successfully to obtain numerous protein-protein conjugates, they possess a major inherent deficiency; it is impossible to determine the amount of incorporation of crosslinkers, e.g. degree of activation of a protein with SMCC. In addition, some reactive groups, e.g. maleimide or NHS can undergo rapid hydrolysis, which makes precise determination of the incorporation of a crosslinker into the activated intermediate impossible.
Therefore, there is a need in the field for bis-heterofunctional crosslinking reagents that allow for very quick, non-destructive determination of crosslinker incorporation into an activated peptide biopolymer. The extensive use of avidin/biotin technology in immunology and diagnostic medicine is based on numerous factors. The high binding affinity between avidin and biotin yields an avidin-biotin complex having very high stability. Avidin is a tetrameric glycoprotein made up of four identical subunits. Therefore, four biotin molecules are capable of binding to one avidin molecule, Adv. Prot. Chem. 29:85 (Green, N. M., 1975), thereby increasing the sensitivity of constructed probe systems, and allowing the crosslinking of two or more biotin-labeled materials.
The disassociation constant for the avidin-biotin complex is 10−15 at neutral pH, Biochem. J., 89:585 (Green, N. M., 1963). This interaction is one of the strongest non-covalent associations found in nature. Biotin is deeply bound in a groove in the avidin molecule with the carboxyl group about 9 angstroms below the protein surface, Biochem. J., 125:781 (Green, N. M., et al., 1971). The widespread use of biotin-avidin technology is based upon the fact that a biotin molecule is easily coupled via a covalent linkage to a protein, while maintaining substantially all the biological properties of the protein and the binding capacity of biotin to avidin.
The extremely high binding constant and fast kinetics of binding and the stability of avidin under a variety of conditions make this an ideal ligand/receptor pair for these purposes. Biotin has been modified to include amino, thiol and carbohydrate reactive moieties, i.e. succinimidyl ester, maleimido and hydrazide respectively, to allow easy incorporation into a large variety of biomolecules. To accomplish detection of an analyte, biotin is conjugated to a probing biomolecule, such as an antibody or an oligonucleotide. Following binding of the biotinylated biomolecule to its receptor or complement, an avidin/reporter conjugate, such as an avidin/fluorophore conjugate or a avidin/reporter enzyme conjugate is added and allowed to bind to a biotinylated probe and visualized by fluorescence detection or addition of a substrate that emits light or precipitates a colored insoluble product on enzymatic processing (Heitzmann H., Richards F. M., Proc. Natl. Acad. Sci. USA 71:3537-3541, 1974; Diamandis E. P., Christopoulos T. K., Clin. Chem. 37:625-636, 1991; Wilchek M. Methods Enzymol Vol. 184, 1990; Savage, M. D. et al., 1992 Avidin-Biotin Chemistry: A Handbook. Rockford, Ill.: Pierce Chemical Co.).
Following conjugation, it is important to confirm that the probe molecule has been biotinylated and to quantify the number of biotins conjugated to the probe molecule. To this end two multi-step indirect assays have been developed. The first assay is the HABA ([2-(4′-hydroxyazobenzene)]benzoic acid) assay developed by Green (Green, N. M. Biochem. J., 94, 23c-24, 1965). To quantify biotin label incorporation, a solution containing the biotinylated protein is added to a mixture of HABA and avidin. Because of its higher affinity for avidin, biotin displaces the HABA from its interaction with avidin and the absorption at 500 nm decreases proportionately. By this method, an unknown amount of biotin present in a solution can be evaluated in a single cuvette by measuring the absorbance of the HABA-avidin solution before and after addition of the biotin-containing sample. The change in absorbance relates to the amount of biotin in the sample.
The second more sensitive fluorescence-based multi-step assay developed by Molecular Probes is the ‘Fluoreporter Biotin Quantitation Assay’ that is based on fluorescence resonance energy transfer (FRET) quenching wherein an avidin molecule is labeled with a fluorophore and its binding sites are occupied with a fluorescent molecule that quenches the covalently linked fluorophore until the quencher in the binding site is displaced by a higher binding biotin molecule resulting in fluorescence of the covalently attached fluorophore. While this assay is sensitive to 50-100 .rho.mol range it requires many processing steps and a fluorimeter or multi-well fluorimeter. It is also recommended that the biotinylated protein be digested prior to the assay to expose any sterically encumbered biotins.
The direct method of hapten incorporation developed by Solulink Inc (US Pat Pub 20080221343) is based on incorporation of hydrazone-based, spectrophotometrically quantifiable group into a linker. The incorporation of the hapten into a protein or other macromolecule can be detected by non-destructive UV-Vis spectroscopy. While the incorporation of the hydrazone moiety into a linker allows for direct, and non-destructive determination of degree of incorporation, it possess a major disadvantage with regard to the long term stability of the hydrozone moiety. Under certain conditions the hydrozone bond can undergo hydrolysis resulting in dissociation of hapten from analyte Angew. Chem. Int. Ed, 49:11 (Anouk, D., et al., 2010).
Consequently there is a need in the field for a assay wherein the number of biotins covalently linked to a biomolecule con be determined by direct methods, such as spectroscopic means, without compromising of long-time stability of conjugates.